1. Field of the Invention
The invention is generally related to the field of detecting and identifying genetic materials. such as nucleic acid molecules of interest, using a nucleic acid detector. The nucleic acid detector according to the invention is constructed from double stranded hybridization probes that are immobilized on a support, to form a DNA detector. The DNA detector is formed into an array of varied binding specificities that is capable of detecting multiple genetic loci. The probes are oligonucleotides each comprising a hypervariable number of tandem repeats ("VNTR") that are anchored at one terminus to a support and at the other terminus to a reporter moiety.
2. Description of the Related Art
Previously, the best method known for detecting and identifying a nucleic acid analyte (e.g., an unknown sample) has been so-called DNA fingerprinting, which relies on a comparison of the electrophoretic migration of restriction fragments of an unknown nucleic acid analyte to the electrophoretic migration pattern of a known genomic sample subjected to identical restriction treatment. This process is typically, but not exclusively, employed to identify or detect deoxyribonucleic acid ("DNA"). Variations on this process are known, including the use of specific hybridization probes to enhance accuracy by confirming that migration bands are homologous to specific nucleic acid sequences of interest. This is possible because restriction enzymes cleave DNA at specific loci, which will vary (i.e., exhibit polymorphism) with each genome. Thus, when DNA gel banding techniques where developed, it seemed possible that the technique would provide unique and unequivocal comparisons and identification between genomic samples.
DNA fingerprinting has been relied upon to analyze forensic evidence, for example, to obtain evidence of the identity of genetic material for criminal or paternity proceedings. The technique is also used to identify human remains or to determine species relatedness. DNA fingerprinting using VNTR loci complementary probes is useful not only in a forensic laboratory setting to provide individual identification and paternity testing, but also has been extensively used for the investigation of taxonomic relationship among fish, birds, plants, wild and domestic animals. In addition, it has also been used for clarifying genetic relationship among related species, for discriminating pathogens from non-pathogens, and for determining the effect of environmental factors on evolutionary dynamics and speciation of microorganisms.
Of course, as the artisan will appreciate, the accuracy of the DNA fragment size-sharing principle has been the subject of criticism e.g., in numerous legal cases, because similarities in the electrophoretic mobility, e.g., size, of restriction fragments on DNA electrophoretic profiles does not unequivocally establish the full characteristics of the alleles.
Therefore, greater accuracy was required before DNA gel banding techniques would be accepted as an accurate means of genetic profiling. While the classical genetic markers. such as the single-locus probe (SLP), have been employed in an attempt to provide improved identification. these usually give little information about the individualization of species due to the very large number of SLP's necessary to determine the exact relationship between species. The use of multiple SLPs for identification purposes has been known as "multiplexing.".sup.15
In 1985, geneticist Alec Jeffreys (Jeffreys, et al., 1985, Nature 314:67-73) partially solved the accuracy problem by applying certain specific hybridization probes to electrophoresis patterns produced by DNA restriction fragments. Since then. while DNA profiling has become an accepted scientific and forensic tool, a great deal of public controversy has continued to be generated by these methods, particularly when used for forensic identifications.
The method described by Jeffreys et al., supra, was based on DNA gel banding techniques with the added feature of detecting polymorphisms in minisatellite DNA. The term "satellite" was originally derived from the observation that DNA isolated from eukaryotes under buoyant density gradient ultracentrifugation, shows extra peaks beside the major DNA bands (Bretton. R J et al., 1968. Science 161: 529-540). Satellites are relatively large chromosomal structures which contain millions of repetitive sequences. Similar repetitive sequences were later found to be almost universally present in the genomic structures of most eukaryotes, as well as in genome viruses. These sequences were called "minisatellites", "midisatellites", and "microsatellites" because of their limited degrees of repetition, i.e., small size (Jeffreys et al., 1985, supra) relative to "satellites", which contain millions of repetitive sequences.
For example, the term, "minisatellite" is applied to any of a class of dispersed arrays of short (e.g., 10-50 bp) tandem direct repeat motifs that contain variants of a common core sequence (e.g., 10-15 bp). The majority of minisatellites are distributed at the terminal ends of genomes. Although their exact functions are not clearly known these terminal repeats may play a significant role in replication control of genes. The major difference of TR's from classical genetic markers is in the hypervariable number of the tandem repeats (VNTR). The VNTR therefore provides extremely useful information about relatedness and individualization of species and other types of nucleic acid analytes.
The human-derived minisatellites such as 33.6,33.15, MS1, CMM1O 1YNH24, EFDS2, TBQ7, MS43 and JE46 are commonly used to prepare hybridization probes for forensic testing, for example, to provide individual identification and paternity testing. These minisatellites can also hybridize to DNA isolated from avians, plants, fish, and other mammals.sup.3-6, thus indicating the presence of common genetic structures in these living organisms.
The multiloci human probes 33.6, 33.15, and MS1 have been used for fingerprinting of DNA isolated from pigs, mice, and common marmosets,.sup.3 for characterization of genetic relationship between breedings of poultry, and for studying a population of foxes in the California Channel Islands..sup.8 Application of the multilocus fingerprinting probe (MLP) 33.15 also revealed genetic profiles in species and strains of Leishmania and Trypanosoma cruzi..sup.9 Fingerprinting data can permit the construction of pedigrees that reflect the population history and the geographical distance of different species and strains of the parasites.
Another type of abundantly distributed repetitive sequences are the microsatellites, which have an even smaller degree of repetition than that of the minisatellites. They are distinguishable from minisatellites by having repetition within the repetitive units. The majority of minisatellites are usually distributed at the terminal ends of genes, while microsatellites are widespread along the chromosomes. Microsatellites used extensively for DNA-fingerprinting have the general structural characteristics of (CA)8, (CT)8, (CAC)5, (GAC)5, (GACA)4, (GATA)4, and the like. They are abundantly distributed in genomic structures of living organisms. Dinucleotide repeats, particularly CA/GT repeats, are very abundant and polymorphic. In other words, they are extremely variable in number of the repeat units. .sup.13 Using (GTG), (GACA) or phage M13 core sequence as either hybridization probes or primers in combination with restriction enzymes with a recognition site of 6 base pairs (bp), over 70 species representing 18 genera filamentous fungi and 5 genera of yeasts were fingerprinted, and their DNA banding patterns and taxonomic relationship were clearly identified. .sup.14 These ubiquitously interspersed, tandemly repetitive sequences with a total number of bases ranging from 2 to 6 are highly polymorphic, rendering these simple short tandemly repeated sequences superior to other polymorphic sequences for individual and species identification purposes. Results obtained from DNA-fingerprinting using either minisatellites or microsatellites agree well with traditional taxonomic classification based on morphological characteristics.
Minisatellite and microsatellite DNA probing of DNA fragments has advantages over conventional restriction fragment length DNA-polymorphism. It is relatively rapid, simple, and reliable, as well as more easily applicable to large scale experiments. Application of VNTR sequences as complementary probes for DNA gel banding techniques yields electrophoretic patterns substantially unique to each individual and species or strain. As for the processes conducted with conventional probes, these assays are also commonly referred to "DNA fingerprinting."
Essentially, genomic material or forensic material believed to contain genomic material is subjected to cleavage by one or more restriction enzymes as discussed above. Electrophoretic separation of the restriction fragments followed by contact with labeled VNTR probes specifically complementary to specific regions of the cleaved genomic DNA produces the DNA profile or fingerprint.
If, for example, two samples of genetic material produce sufficiently matching DNA gel banding and VNTR labeling patterns (i.e., the two samples cleave into fragments of the same size, migrating the same distance on the gel, and have homologous regions complementary to the same VNTR loci), the conclusion is that the samples are from the same source, to a high degree of probability.
According to Chakrabarty and Jin,.sup.19 more than 50 SLP's may be needed for positive identification of an individual or species. A single SLP could only detect a piece of DNA sequence among huge genomes of living cells, yielding little information as to what has been detected. In comparison, when multi-locus probes ("MLP") such as minisatellites or microsatellites are used, it is estimated that only about 7 minisatellites and 15 microsatellites may be required for positive individual or species identification. For simultaneous typing with MLP, a panel of nucleic acid detector molecules are constructed (exemplified by Table 1, presented hereinbelow) according to the known information of VNTR's of the particular species to be studied. Binding patterns as well as the binding characteristics of each member of the panel can be monitored simultaneously. When nucleic acid found in an analyte specimen is considered to be identical to that obtained from an individual, all patterns and characteristics of bindings must match each other well for positive identification. Relatedness among individuals and species can be also calculated from the similarity index(x) or "alleles sharing coefficient" (D) according to the total number of the shared alleles and total number of alleles present from data obtained from a panel of binding assays.
Statistical Considerations Of Relatedness Among Individuals And Species
The basic principle for application of allele sharing for individual/species and relatedness determination is based on the number of loci and the number of shared alleles of genotypes of individual entities. For quantifying the differences between two DNA fingerprinting profiles, "band-sharing coefficients" (D) or similarity indices (x) have been used, and the relationship between the two individuals can be estimated according to the following equations (according to references 1, 16, 17, 18 and 19, provided hereinbelow). EQU D=2Nab/(Na+Nb) (1) EQU x=((Nab/Na)+(Nab/Nb))/2 (2).
Nab is the number of scorable bands to two DNA profiles A and B. Na is the number of scorable bands in A. Nb is the number of scorable bands in B. The band-sharing values obtained from the above equations yield similar results. In humans, band-sharing coefficient, D or similarity index x is about 0.2 for a pair of unrelated individuals..sup.16 The average values could reach 0.8 between siblings and between parents and offspring..sup.17-18 In plants, the average band-sharing coefficient can reach 0.5 due to vegetative method of propagation. When in-breeding rate is high among those populations, D or x values are also high. For DNA fingerprints derived from the same individual, as in the cases of forensic specimen analysis, Nab=Nb=Na, and the D or x value is equal to one.
Thus, from a statistical point of view, the application of the VNTR loci to conventional DNA fingerprinting methods, by providing a multiplicity of loci for comparison. has improved the accuracy and reliability of DNA profiling by enhancing confidence in the significance of bands of the same size in two samples under comparison.
However, problems remain. It has been argued in numerous forensic legal proceedings that DNA fragments that have same sizes, although they migrate to the same positions in agarose gel, are not necessarily identical in their actual sequences unless all fragments of the electrophoresis profiles are measured. Heretofore, sequence analysis of multiple DNA fingerprint bands to produce unequivocal identification data has been deemed impractical because of the tedious, time-consuming, and costly procedures involved in such DNA sequencing.
Although MLP's reduce the total number of probes required for reliable identification, there remains the requirement for electrophoretic separation of genomic fragments before such MLP probes can be applied to DNA fingerprinting methods. In addition, the previously employed DNA fingerprinting technique described above, even when conducted using VNTR probes, remains a tedious and time consuming process requiring sophisticated laboratory facilities. Further. the technique must be performed by an individual with extensive training in molecular biology and chemistry and certainly cannot readily be practiced under field conditions, e.g., outside of a laboratory.
Therefore, it is clear that alternative methods for rapid, accurate and simple methods for biological detection or identification of nucleic acid analytes are required. The art has attempted to provide alternative techniques. For example, one art-known alternative to gel banding techniques is the use of non-gel capillary electrophoresis and the nuclei condensation electrospray to provide size separation and fractionation of macromolecules..sup.20 However, this technique can only provide information about the size of each DNA fragment obtained from restriction digest. Since no detailed sequence information can be derived from this procedure. the problem of distinguishing differing genomic fragments, which nevertheless have the same measured molecular weight, remains unresolved. Moreover. as for the agarose gel electrophoresis previously described, application of sample to the non-gel capillary is also challenging and requires highly skilled practitioners.
It is therefore clear that. despite the foregoing efforts, there remains a need in the art for new approaches to nucleic acid profiling that are applicable for initial screening purposes for large numbers of samples (e.g., species and individuals), able to be conducted without sophisticated instrumentation. For example, there is a need for nucleic acid profiling methods that avoid the use of electrophoresis and can be conducted, for example, under field, or even battlefield, conditions, in order to obtain reliable identification or characterization of genomic material (e.g., identification of pathogen. animal and/or human tissue samples). In particular, it would be desirable to have available a DNA/RNA detection method that does not require elaborate and complex processes to conduct prehybridization. hybridization. linkage, blottings (Southern or Northern), electrophoresis and other manipulation steps.